FIG. 5.

Crkl is essential for Fgf8-induced activation of Rac1 and Cdc42, but not Ras. (A) Pulldown assays for GTP-bound small G proteins. Wild-type MEFs were stimulated with either Fgf8 or EGF, while cells were kept in suspension. Cell lysates were prepared after 10 min of stimulation. The levels of total Ras, Rac1, and Cdc42 are shown in the lower panel for each GTP-bound G protein pulldown. (B) Pulldown assays for GTP-bound small G proteins after 10 min of stimulation with 10 ng of Fgf8/ml in suspension culture of MEFs isolated from wild-type or Crkl^−/− embryos. An additional control was prepared by introducing a CRKL transgene into Crkl^−/− MEFs (Crkl^−/− + CRKL). A pulldown assay of GTP-bound small G-proteins was carried out as described above. (C) Coimmunoprecipitation of Fgfr1 with Dock1. Cell lysates were prepared as described above to immunoprecipitate Fgfr1. Dock1 protein was probed in the immunoprecipitate by immunoblotting.